The long-term objectives of this proposal are to understand the fundamental mechanisms by which human hepatitis B virus (HBV) regulates gene expression and contributes to the production of hepatocellular carcinoma (HCC). The specific aims of this proposal can be summarized as four main goals: (1) To precisely map the X protein domains involved in activation and repression of transcription by RNA polymerases II and III. (2) To investigate the role of X protein in regulating transcription and identify potential regulatory target proteins. (3) To investigate whether X protein activates transcription elements by acting directly at the promoter, whether it act on the central regulators of key signal transduction pathways, or both. (4) To identify cellular polypeptides that likely interact with X protein, some of which could be central mediators of transcriptional regulation and cell growth. The manner by which HBV participates in generation of HCC, the factors which contribute to liver disease during chronic infection and the ability of the virus to maintain persistent infections for considerable periods of time are all areas which are poorly understood. The transcriptional regulatory properties of the HBV X protein has therefore been potentially implicated in many of these processes. Experiments have been proposed to investigate the molecular level at which X proteins function and their mechanism of action. Previous studies demonstrated that X protein activates transcription by RNA polymerases II and III. Recent studies indicate that the X gene actually encodes two related polypeptides, a full- length translation product and an amino-truncated species. Experiments are outlined to fully characterize the multiple transcriptional activities of both X proteins (activation and repression), and to finely map the protein regions involved in activation of RNA polymerases II and III. To dissect the molecular level and key cellular proteins acted on by X protein, inducible and regulatable X protein constructs are described which will enable determination of the transcription at the level of the promoter, or through participation in signal transduction pathways, or both. Given the ability to modulate the transcriptional activity of different classes and types of promoters, and the apparent inability to bind DNA, it seems likely that the X proteins function by interacting with important cellular regulatory polypeptides. Several approaches have been outlined to investigate the potential association of X proteins with cellular polypeptides, including identification by co-immunoprecipitation. Attempts will also be made to clone interacting cellular polypeptides by using purified labeled X protein to screen a cDNA expression library prepared from human liver.